The microtubule-associated protein tau is hyperphosphorylated in Alzheimer's disease and related neurodegenerative disorders. Despite accumulating circumstantial evidence implicating a causative role for tau hyperphosphorylation in neurodegeneration, the mechanisms leading to neuronal cell death remain undefined. Proline-directed protein kinases phosphorylate tau on serine (S) or threonine (T) residues that are immediately flanked by proline (P), sequences known as SP or TP motifs. Phosphorylation of tau at SP/TP sites correlates specifically with human disease. In addition to phosphorylation, the production of calpain-cleaved tau fragments has been implicated as a potential source of neurotoxicity. This proposal addresses the hypothesis that phosphorylation regulates the production of proteolytic tau fragments, and that calpain-mediated production of these fragments promotes tau toxicity in vivo. The first specific aim will determine if phosphorylation at SP/TP sites is required for tau to be a substrate for calpain digestion. To determine the phosphorylation requirement, we will test whether an SP/TP-phosphorylation-incompetent form of tau can be cleaved in a Drosophila S2 cell culture system as well as in an in vitro digestion assay. Examination of the subcellular localization of tau and calpain will demonstrate the importance of their interaction at the molecular level. The second specific aim addresses the pathological significance of calpain cleavage of tau in an intact animal model of human neurodegenerative disease. These experiments will test the hypothesis that tau toxicity and the production of tau fragments can be enhanced and suppressed by genetically and pharmacologically modifying calpain. To further investigate the pathological significance of tau fragment production in vivo, a calpain-resistant form of tau as well as the putative calpain-cleaved 17kD tau fragment will be created for expression in Drosophila. The research described in this proposal investigates the molecular pathways involved in Alzheimer's disease using both molecular and genetic approaches. The ultimate goal of this research is to understand the underlying causes of Alzheimer's disease to extend healthy life and reduce the burdens of illness and disability. Elucidating the role that tau phosphorylation and proteolysis play in neurodegeneration may lead to new treatment strategies. PUBLIC HEALTH RELEVANCE: This proposal will investigate the underlying causes of Alzheimer's disease. Understanding the events that cause neuronal cells to die may eventually lead to the development of new therapeutic strategies for preventing or curing Alzheimer's disease. [unreadable] [unreadable]